Ubiquitination is a posttranslational modification that instigates proteasomal degradation of its target proteins. In recent years, the function of ubiquitination has expanded to include roles in membrane trafficking, protein-protein interactions, gene transcription, and kinase activation. Three enzymes mediate ubiquitination: the E1 ubiquitin-activating enzyme, the E2 ubiquitin-conjugating enzyme, and the E3 ubiquitin ligase, which determines the specificity of ubiquitination. Mutations of Skp1/Cullin/F-box E3 ubiquitin ligase complexes (SCF) have been linked to many human diseases, including multiple forms of cancer, immune disorders such as diabetes and lupus, neurological disorders like Parkinson's disease, viral infections, and cardiac myopathies. Our lab has recently demonstrated that a functional SCF complex is required for proper pheromone response in the budding yeast S. cerevisiae. The yeast pheromone response pathway is one of the best-characterized signal transduction pathways, consisting of a canonical G-protein coupled receptor (GPCR), heterotrimeric G- protein, and mitogen-activated protein kinase (MAPK) signaling cascade. Moreover, this signaling pathway has been used to identify multiple cell signaling mechanisms that are conserved in virtually all eukaryotes. We and others have demonstrated that the SCF complex is required for ubiquitination of multiple signaling components within the pheromone response pathway, including the G1 subunit Gpa1, the MAPK kinase Ste7, the scaffold protein Ste5, and the effector Far1. Further, we have recently generated data in our lab demonstrating that ubiquitination alters the selectivity of Ste7 activation of the downstream MAPKs Fus3 and Kss1. Based on these data, we hypothesize that SCF-mediated ubiquitination of Ste7 regulates its ability to selectively activate downstream signaling proteins. We further hypothesize that SCF is a master regulator of the MAPK signaling cascade, acting simultaneously on multiple pathway components to dictate overall pathway output. This hypothesis will be addressed through the following specific aims: Aim #1: Determine how ubiquitination modulates the activity and specificity of the pheromone pathway MAPKK Ste7. Aim #2: Systematically identify all proteins in the pheromone response pathway that are ubiquitinated by the SCF E3 ubiquitin ligase complex. The proposed studies will define how ubiquitination influences the signaling behavior of a target protein, determine the contribution of ubiquitination to overall signaling pathway output, and expand our understanding of how the highly-conserved MAPK cascade is regulated. PUBLIC HEALTH RELEVANCE: Cellular signaling pathways regulate how cells respond to their environment. Incorrect cellular signaling causes many human diseases including cancer, immune disorders like diabetes and lupus, and neurological disorders such as Parkinson's disease. This proposal aims to identify a new mechanism that regulates cellular signaling and possibly reveal new ways to modulate signaling pathways to combat disease.